Prions are proteins devoid of nucleic acids and cell membranes which are largely unaffected by standard methods of sterilisation. Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383). Inactivation of prions poses significant environmental and health issues both for the disposal of prions infected animals and in the preparation of materials of animal origin, such as animal feed. Slaughterhouse sludge and animal mortalities (carcasses) are an important source of pathogens or infectious prions proteins.
Prions are protein naturally found in animals. Cellular or normal form of prion proteins are constitutively expressed in the brains of healthy adult animals, but are highly regulated, both spatially and temporally, during development (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383). There are two isoforms of prion proteins. PrPC (Prion Protein Cellular isoform) is a cell surface, N-linked, α-helices-rich, globular soluble glycoprotein protein that may serve as a signal transduction protein (Mouillet-Richard et al., 2000, Science, 289: 1925-1928) and may play an essential role in the normal development of mammal brain. PrPC occurs both in healthy and diseased tissues. PrPSc (Prion Protein Scrapie agent) is a β-sheet-rich, fibrous, highly insoluble protein which is the causative agent of central nervous system diseases in mammals. It is thought that a higher content in β-sheet confer heat- and protease-resistance to PrPSc.
The presence of the abnormal, pathological isoform (PrPSc) is typical of the diseased state. PrPC and PrPSc are identical in their primary structure. Differences occur in secondary structures: PrPC contains three α-helices (about 40% α-helix) and a short antiparallel β-sheet, whereas PrPSc is composed of two α-helices (about 30% α-helix) and four β-sheets (45% β-sheets) (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383)). This induces conformational changes in the tertiary structure of PrPSc, resulting in modified characteristics. Both PrP isoforms are devoid of nucleic acids (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383).
Experimental data suggests that prions can survive for a long time in natural environments. For example, Brown and Gajdusek (1991, J Infect Dis, 153: 1145-1148) showed that prions buried into a garden soil could survive and retain their infectivity power for 3 years, with little leaching deeper into the soil.
Current research suggests that the primary method of infection in animals is through ingestion. It is thought that prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding to clay and other minerals. Sterilizing prions involves the denaturation of the protein to a state where the molecule is no longer able to induce the abnormal folding of normal proteins. However, prions are generally quite resistant to proteases, heat, radiation, and formalin treatments, although their infectivity can be reduced by such treatments. Effective prion decontamination relies upon protein hydrolysis or reduction and/or destruction of protein tertiary structure. Examples include bleach, caustic soda, or strong acidic detergents.
Transmissible Degenerative Encephalopathies. (TDE) forms a group of fatal neurodegenerative disorders caused by the accumulation of prions in the brains of mammals. TDE are unique in that the host's normal prion protein (PrPc) is modified into the infective prion protein (PrPsc) as a consequence of infection (Carp et al., 1985, J Gen Virol, 66: 1357-1368), and forms deposits in affected tissues, especially in the central nervous system. TDE affect a wide variety of wild animals and livestock, as well as humans, and present in 3 ways, all of which involve modifications of the prion protein: heritably as a result of genetic mutations; sporadically by spontaneous conversion of the prion protein into a pathologic form via yet undefined mechanisms; by infection following exposure to the exogenous misfolded form of the prion protein.
Bovine Spongiform Encephalopathy (BSE) is among the most notable prion disease. The International Trade Commission (ITC) released a report estimating that trade restrictions resulting from Bovine BSE cost the cattle industry $11 billion from 2004 to 2007.
Thus, not only are animal manure management practices are often detrimental to the environment, they also represent a potential hazard to human and animal health, in addition in producing strong odours, encourage fly breeding, induce weed problems and pollute air, soil and water.
The Canadian Food Inspection Agency estimated the amount of specified risk materials (SRM) generated in Canada at 170,000 tonnes annually. Safe disposal of SRM potentially contaminated with prions is challenging since these TDE-causing agents are relatively resistant to inactivation by physical or chemical procedures usually applied for microorganisms (Taylor et al., 1994, Arch Virol, 139: 313-326). Moreover, significant costs are associated with some disposal treatment. Physical and chemical methods of prions inactivation that efficiently and/or rapidly fix proteins, including alcohols, aldehydes and rapid heating with steam, protect prions from inactivation, hence enhancing their thermo stability. Thus, treatments that disrupt protein structure, rather than fixing it, are required when considering the disposal of SRM potentially contaminated with prions. Moreover, treatments using chemicals such as denaturants, detergents, strong alkali are inappropriate for the inactivation of prions in SRM due to potential user exposure at the farm and disposal problems onto agricultural land.
Consequently, there is a need to develop biological treatments for the degradation of prions in SRM which are environmentally sound.
It would be thus highly desirable to be provided with a process that eliminates prion protein in specified risk materials from animal that is low in cost, is very stable, simple, easy to operate and which does not interfere with regular farm operations.